Dyes in general, and fluorescent dyes in particular, are often used to directly stain or label a sample so that the sample can be identified or quantitated. For instance, such dyes may be added as part of an assay for a biological target analyte, as a detectable tracer element in a biological or non-biological fluid, or for such purposes as photodynamic therapy of tumors, in which a dyed sample is irradiated to selectively destroy tumor cells and tissues, or to photoablate arterial plaque or cells, usually through the photosensitized production of singlet oxygen.
Fluorescent dyes with longer wavelength absorption and emission are particularly useful in conjunction with materials of biological origin such as blood, urine, fecal matter, cells and tissues, where background or inherent fluorescence or absorption often interferes with detection of the added fluorescent dye. Furthermore, biological specimens often have decreasing levels of both absorption and fluorescence emission as the illumination energy approaches the infrared. In addition, numerous biological and nonbiological applications of long wavelength dyes exist, including use as laser dyes, or in electronics as optical memory elements using relatively low cost illumination sources such as laser diodes. Consequently, dyes that possess these spectral properties have potential utility in biological and non-biological applications.
A variety of dipyrrometheneboron difluoride dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes) have previously been described, by these inventors, and others, in which substituents are attached by a single covalent bond, including U.S. Pat. No. 4,774,339 to Haugland et al. and U.S. Pat. No. 5,274,113 to Kang et al. (reactive substituents), U.S. Pat. No. 5,248,782 to Haugland et al. (heteroaryl substituents), U.S. Pat. No. 5,187,288 to Kang et al. (ethenyl substituents), and U.S. Pat. No. 5,338,854 to Kang et al. (fatty acid substituents). All of the above patents describe fluorescent dyes and conjugates of fluorescent dyes based on the dipyrrometheneboron difluoride core structure shown below: ##STR2##
In addition, dipyrrometheneboron difluoride dyes incorporating fused aromatic rings (dibenzopyrrometheneboron difluoride dyes, structure shown below) have been described by Kang et al. (U.S. Pat. No. 5,433,896 (1995); incorporated by reference). The dibenzopyrrometheneboron difluoride dyes possess longer wavelengths than other, previously described dipyrrometheneboron difluoride dyes. ##STR3##
The dyes of the present invention typically possess even longer wavelength excitation and emission bands than previously described dipyrrometheneboron difluoride dyes, including dibenzopyrrometheneboron difluoride dyes. Like the dibenzopyrrometheneboron difluoride dyes, the spectral properties of the dyes of the present invention are substantially insensitive to the chemical environment of the dye, even when compared to the previously described dipyrrometheneboron difluoride dyes. Practically this means that the absorbance and emission of the dyes differ only slightly when measured in different solvents. The subject dyes are relatively stable to photobleaching, remaining intensely fluorescent even when constantly illuminated. Finally, the dyes of the present invention exhibit very long wavelength excitation and emission bands, thereby showing enhanced utility in sample systems that possess transparency primarily in the infrared region, or where the use of infrared wavelengths is particularly advantageous.